Method and system for overlaying at least three microarray images to obtain a multicolor composite image

ABSTRACT

Method and system for overlaying at least three microarray images to obtain a multicolor composite image, which is then displayed on a monitor of a computer system. The microarray images are taken from a microarray scanner of a DNA microarray and can be viewed simultaneously through the use of the image overlays where each image is represented by a different color. Each pixel of the composite image is generated by the OR operator applied to all corresponding pixels of the microarray images. Registration of the microarray images can be altered with a keyboard or mouse of the computer system.

TECHNICAL FIELD

This invention relates to methods and systems for overlaying at leastthree microarray images to obtain a multicolor composite image.

BACKGROUND ART

Spots containing fluorescently labeled DNA samples on a suitable carriersuch as a microscope slide or membrane are commonly known as microarraysor bio-chips. An example of a process that uses overlays is in theanalysis and quantitation of such microarrays.

The microarrays are typically created with fluorescently labeled DNAsamples in a grid pattern consisting of rows 22 and columns 20 typicallyspread across a 1 by 3 inch glass microscope slide 24, as illustrated inFIG. 1. The rows 22 extend along the smaller dimension of the slide 24and the columns 20 extend along the larger dimension of the rectangularslide 24. Each spot 26 in the grid pattern (or array) 28 represents aseparate DNA sample and constitutes a separate experiment. A pluralityof such grid patterns 28 comprises an array set 30. Reference or“target” DNA (or RNA) is spotted onto the glass slide 24 and chemicallybonded to its surface. Fluorescently labeled “probe” DNA (or RNA) isthen introduced and allowed to hybridize with the target DNA. Excessprobe DNA that does not bind is removed from the surface of the slide 24in a subsequent washing process.

The purpose of the experiment is to measure the binding affinity betweenthe probe and target DNA to determine the likeness of their molecularstructures: complementary molecules have a much greater probability ofbinding than unrelated molecules. The probe DNA is labeled withfluorescent labels that emit light when excited by an external lightsource of the proper wavelength. The brightness of each sample on theslide 24 is a function of the fluor density in that sample. The fluordensity is a function of the binding affinity or likeness of the probemolecule to the target molecule. Therefore, the brightness of eachsample can be mapped to the degree of similarity between the probe DNAand the target DNA in that sample. On a typical microarray, up to tensof thousands of experiments can be performed simultaneously on the probeDNA, allowing for a detailed characterization of complex molecules.

Scanning laser fluorescence microscopes or microarray readers asillustrated in FIG. 2 can be used to acquire digital images of theemitted light from a microarray. The digital images are comprised ofseveral thousand to hundreds of millions of pixels that typically rangein size from 5 to 50 microns. Each pixel in the digital image istypically represented by a 16 bit integer, allowing for 65,535 differentgrayscale values. The microarray reader sequentially acquires the pixelsfrom the scanned microarray and writes them into an image file which isstored on a computer hard drive. The microarrays can contain severaldifferent fluorescently tagged probe DNA samples at each spot location.The microarray scanner repeatedly scans the entire microarray with alaser of the appropriate wavelength to excite each of the probe DNAsamples and store them in their separate image files. The image filesare analyzed and subsequently viewed with the aid of a programmedcomputer.

A typical confocal laser microarray scanner or microarray reader isillustrated in FIG. 2. The reader is commonly used to scan themicroarray slide 24 to produce one image for each dye used bysequentially scanning the microarray with a laser of a proper wavelengthfor the particular dye. Each dye has a known excitation spectra and aknown emission spectra. The scanner includes a beam splitter 32 whichreflects a laser beam 34 towards an objective lens 36 which, in turn,focuses the beam at the surface of slide 24 to cause fluorescencespherical emission. A portion of the emission travels back through thelens 36 and the beam splitter 32. After traveling through the beamsplitter 32, the fluorescence beam is reflected by a mirror 38, travelsthrough an emission filter 40, a focusing detector lens 42 and a centralpinhole 44.

Analysis and Quantitation of the Microarray in Two Colors

Analysis of the fluor density at each spot location requires softwarealgorithms that utilize image processing algorithms to locate all thespots and measure the brightness of the pixels in each spot. Visualfeedback of the relative spot intensities has commonly been done usingsoftware to overlay the two images by encoding the brightness of theimage as a function of the brightness of one color for each image, i.e.red or green. When red and green objects are overlayed at the same pixellocation, a yellow color is produced as illustrated in FIG. 3.

Given

B1 is the brightness of a pixel in image 1,

B2 is the brightness of a pixel in image 2,

each brightness value is from 0 to 65,535,

Image 1 is to be overlayed in red and an 8 bit pixel will be R1,

Image 2 is to be overlayed in green and an 8 bit pixel will be G1.

R1=B1/256

G1=B2/256

A common method for displaying pixels on a computer monitor or printoutis in true color, comprised of 24 bits per pixel, where 8 bits eachrepresent the red, green, and blue color channels, respectively.

Pixel (R, G, B)=R 1, G 1, 0

The visual display of the overlayed images provides the viewer withfeedback on the brightness of each spot from each channel of the reader,the relative brightness of each spot to the others by the degree of thecolor yellow present, and a measure of the spatial registration betweenimages by the amount of red or green on either side of a central yellowcolor as illustrated in FIG. 3.

The use of two color overlays is common and has been demonstrated byBioDiscovery in a software program called ImaGene and by StanfordUniversity in a software program called ScanAlyze, as well as severalplaces in the literature. Two color visualization has been designed tomeet the needs of two color microarray experiments. Two color microarrayreaders have been designed and built by several companies and arereadily available in the market. Microarray readers with more than twocolors have just recently been released by the assignee of the presentapplication.

One common use of overlays in microarray experiments and quantitation isto provide visual feedback to scientists of any mis-registration betweenimages. Many microarray readers sequentially scan the microarray withone scan for each DNA probe used. A scan typically starts in the upperleft corner of the microarray, usually called the origin, and proceedsin a left to right and top to bottom direction until the entire area hasbeen scanned. When a scan has finished, the scanner mechanically resetsthe laser to the origin in preparation for the next scan. The differencein the actual position between each image scan is a mis-registrationerror. By viewing the overlayed image and by zooming the spot features,a scientist can visually quantify the amount of mis-registration betweenthe images.

Another common use of the overlays in microarray experiments is tocorrect for any mis-registration errors. Typically, the images arepresented in a single viewing window with each image assigned adifferent color. In a two color system, this would typically be greenand red. The locations where a match is obtained will be displayed inyellow. The scientist can visually see the areas of green and red on thefringes of a microarray spot and, using a keyboard or mouse, move oneimage relative to the other until the fringe areas turn yellow. The bestregistration match will minimize the green and red fringes across theentire microarray pattern.

Articles related to the present invention include the following:

Brown, A. J., et al. “Targeted Display: A New Technique for the Analysisof Differential Gene Expression”, METHODS ENZYMOL. 1999; 303:392-408;

Eisen, M. B., et al. “DNA Arrays for Analysis of Gene Expression”,METHODS ENZYMOL. 1999; 303:179-205;

Zhang, H., et al. “Differential Screening of Gene Expression DifferenceEnriched By Differential Display”, NUCLEIC ACIDS RES. Jun. 15,1996;24(12):2454-5;

Adryan, B., et al. “Digital Image Processing For Rapid Analysis ofDifferentially Expressed Transcripts on High-Density cDNA Arrays”,BIOTECHNIQUES. Jun. 26, 1999(6):1174-9;

Wadler, S., et al. “Quantification of Ribonucleotide ReductaseExpression in Wild-Type and Hydroxyurea-Resistant Cell Lines EmployingIn Situ Reverse Transcriptase Polymerase Chain Reaction and aComputerized Image Analysis System”, ANAL. BIOCHEM. Feb. 1,1999;267(1):24-9; and

Zhao, N., et al. “cDNA Filter Analysis: A Novel Approach ForLarge-Scale, Quantitative Analysis of Gene Expression”, GENE. Apr. 24,1995; 156(2):207-13.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method and system foroverlaying at least three microarray images to obtain a multicolorcomposite image.

In carrying out the above objects and other objects of the presentinvention, a method is provided for overlaying at least three microarrayimages to obtain a multicolor composite image. The method includesproviding at least three microarray images wherein each of the imagesincludes a plurality of pixels having brightness values. The method alsoincludes assigning each image a different color to obtain colorizedimages and combining corresponding pixels of each colorized image toobtain overlayed images. The overlayed images form a multicoloredcomposite image. The method also includes the step of displaying themulticolored composite image.

When the overlayed images have a registration, the method may includethe step of processing the overlayed images to alter the registration.The step of processing may include the step of receiving a command tomove one of the overlayed images relative to the other overlayed images.

The step of providing may include the step of scanning a microarray atat least three different wavelengths to obtain the microarray images.

The step of assigning may include the step of creating a palette havingentries for each of the wavelengths to display the microarray images.The entries for each palette are based on a composite color and thebrightness values of the pixels of its respective image.

Also, preferably, the step of combining includes the step of logicallyORing corresponding pixels of each colorized image.

Still, preferably, the method includes the step of receiving at leastone command to select a composite color for each microarray image to beoverlayed.

Still further in carrying out the above objects and other objects of thepresent invention, a system is provided for carrying out each of theabove method steps.

The above objects and other objects, features, and advantages of thepresent invention are readily apparent from the following detaileddescription of the best mode for carrying out the invention when takenin connection with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a top plan schematic view illustrating a spot, an array and anarray set on a glass slide;

FIG. 2 is a schematic view of a confocal laser reader used to generatedigital images;

FIG. 3 is a schematic view of overlayed red and green objects with acentral yellow color;

FIG. 4 is a block diagram flow chart illustrating the method of thepresent invention; and

FIG. 5 is a schematic diagram illustrating a preferred hardwareconfiguration on which at least a portion of the method of the presentinvention can be implemented.

BEST MODE FOR CARRYING OUT THE INVENTION

Referring now to the drawing figures, there is illustrated in FIG. 5 aworkstation on which the method and system of the present invention canbe implemented. However, other configurations are possible. The hardwareillustrated in FIG. 5 includes a monitor 10 such as a single SVGAdisplay, a keyboard 12, a pointing device such as a mouse 14, a magneticstorage device 16, and a chassis 18 including a CPU and random accessmemory. The monitor 10 may be a touch screen monitor used in addition tostandard keyboard/mouse interaction. In a preferred embodiment, thechassis 18 is a Pentium-based IBM compatible PC or other PC having atleast 32 megabytes of RAM and at least 12 megabytes of hard disk space.The workstation typically includes a Windows NT, graphical userinterface as well as an Ethernet 10 Base-T high speed Lan networkinterface.

Analysis and Ouantitation of the Microarray in More Than Two Colors

Software visualization tools are required to meet the needs of theresearcher using a reader with more than two scanning laser wavelengths.The design of the overlay software algorithms is not trivial when morethan three colors are required due to common practice of representingtrue color as the compilation of three components, red, green, and blue,each of 8 bits, for a 24 bit color. Natural mathematical propertiesexist which easily and clearly combine two or even three colors into anoverlayed true color image. Overlaying more than three colors is not asvisually intuitive.

Microsoft® Windows uses palettes to display all eight bit images. Apalette consists of up to 256 values for each of three different colors,red, green, and blue. The intensity of each 8 bit pixel, with a value of0 to 255, is used to index into the palette. The three values, red,green, and blue, are used to display the pixel in the appropriate colorand intensity.

Referring now to FIG. 4, there is indicated in block diagram flow chartform a method for overlaying and then displaying a multicolor compositeimage from at least three microarray images in accordance with thepresent invention.

At block 50, typically a user selects a composite color for eachmicroarray image to be overlayed using the workstation of FIG. 5.

At block 52, a microarray is scanned such as by the microarray reader orscanner of FIG. 2 at at least three different wavelengths to obtainscanned microarray images.

For each laser wavelength, at block 54, create a palette based on theuser-selected composite color in any combination of red, green and bluevalues. For example, the user can select the color cyan (R=0, G=255,B=255). That chosen color is assigned to the brightest pixel in thescanned image. In most cases this will be 255. The remaining positionsin the palette are successively dimmer representations of the samecolor. The algorithm of block 54 assigns palette entries for one imagebased on “NumEntries” palette entries and the chosen color representedby the combination of three components, “red”, “green” and “blue”.Typical pseudo-code for doing this is as follows:

int red=0, green=255, blue=255, NumEntries=256;

PALETTEENTRY pe[256];

for (int i=0 ; i<NumEntries ; i++)

{

// divide by NumEntries for full spectrum

pe[i] .peRed=(BYTE) ((float) (i*red)/(float)NumEntries);

pe[i] .peGreen=(BYTE) ((float) (i*green)/(float)NumEntries);

pe[i].peBlue=(BYTE) ((float) (i*blue)/(float)NumEntries);

}

pImage−>SetPaletteEntries (0, NumEntries, pe);

This process is repeated for each image scanned by the laser reader andwith a different selected composite color (RGB) as illustrated by block56.

Now that each image has a palette with its chosen composite color, theimages are overlayed. At block 58, for all pixels in a first image,assign each pixel directly to a memory buffer for later drawing ormanipulation.

At block 60, for all pixels in the remaining images, logically “OR” eachpixel with the corresponding pixel in the buffer.

The following is some exemplary pseudo-code for doing this:

for j=0; j<npixels; j++)

{

buf[j]=image[0] [j];

}

for (i=1; i<nimages−1; i++)

{

for (j=0; j<npixels; j++)

{

buf[j]|=images [i] [j];

}

The OR operation used to combine the images is only one of severalBoolean operators that can be used to produce varying results. The useof other Boolean operators and combinations of them to produce othereffects is included. Other standard Boolean operations for combiningimages include XOR, NOT and AND.

At block 62, the computer of FIG. 5 receives a command from the user tomove one of the overlayed images relative to the other overlayed imageto correct for misregistration between the images. This is typicallydone such as by the keyboard 12 and/or the mouse 14 or other computerinterface control.

While embodiments of the invention have been illustrated and described,it is not intended that these embodiments illustrate and describe allpossible forms of the invention. Rather, the words used in thespecification are words of description rather than limitation, and it isunderstood that various changes may be made without departing from thespirit and scope of the invention.

What is claimed is:
 1. A method for overlaying at least three microarrayimages to obtain a multicolor composite image, the method comprising:providing at least three microarray images wherein each of the imagesincludes a plurality of pixels having brightness values; assigning eachimage a different color to obtain colorized images; combiningcorresponding pixels of each colorized image to obtain overlayed imageswhereby the overlayed images form a multicolor composite image; anddisplaying the multicolored composite image.
 2. The method as claimed inclaim 1 wherein the overlayed images have a registration and wherein themethod further comprises processing the overlayed images to alter theregistration of the overlayed images.
 3. The method as claimed in claim2 wherein the step of processing includes the step of receiving acommand to move one of the overlayed images relative to the otheroverlayed images.
 4. The method as claimed in claim 1 wherein the stepof providing includes the step of scanning a microarray at at leastthree different wavelengths to obtain the microarray images.
 5. Themethod as claimed in claim 4 wherein the step of assigning includes thestep of creating a palette having entries for each of the wavelengths todisplay the microarray images, the entries for each palette being basedon a composite color and the brightness values of the pixels of itsrespective image.
 6. The method as claimed in claim 1 wherein the stepof combining includes the step of logically ORing corresponding pixelsof each colorized image.
 7. The method as claimed in claim 1 furthercomprising receiving at least one command to select a composite colorfor each microarray image to be overlayed.
 8. A system for overlaying atleast three microarray images to obtain a multicolor composite image,the system comprising: means for providing at least three microarrayimages wherein each of the images includes a plurality of pixels havingbrightness values; a computer programmed to: assign each image adifferent color to obtain colorized images; and combine correspondingpixels of each colorized image to obtain overlayed images whereby theoverlayed images form a multicolor composite image; and a display fordisplaying the multicolor composite image.
 9. The system as claimed inclaim 8 wherein the overlayed images have a registration and wherein thecomputer is further programmed to process the overlayed images to alterthe registration of the overlayed images.
 10. The system as claimed inclaim 9 wherein the computer is programmed to receive a command to moveone of the overlayed images relative to the other overlayed images. 11.The system as claimed in claim 8 wherein the means for providingincludes a microarray scanner for scanning a microarray at at leastthree different wavelengths to obtain the microarray images.
 12. Thesystem as claimed in claim 11 wherein the computer is programmed tocreate a palette having entries for each of the wavelengths to displaythe microarray images, the entries for each palette being based on acomposite color and the brightness values of the pixels of itsrespective image.
 13. The system as claimed in claim 8 furthercomprising a memory buffer and wherein the computer is furtherprogrammed to assign each pixel in one of the colorized images to thememory buffer and logically ORing corresponding pixels in the remainingcolorized images with the pixels in the memory buffer to obtain theoverlayed images.
 14. The system as claimed in claim 8 wherein thecomputer is further programmed to receive at least one command to selecta composite color for each microarray image to be overlayed.